Protein kinases are enzymes that modify neutral amino acid residues on target proteins to add a charged phosphate group. These enzymes are key regulators of cell function and the DNA encoding their expression constitutes one of the largest and most functionally diverse gene families, constituting approximately 1.7% of the entire human genome. (Manning et al., 2002).
Kinase activity represents one of the most important and prevalent signaling pathways in cells. By adding phosphate groups to substrate proteins, kinase molecules direct the activity, localization, and overall function of many proteins, and serve to orchestrate the activity of almost all cellular processes. Kinases are particularly prominent in signal transduction and co-ordination of complex cellular functions. For example, many proteins are imported into the cell nucleus once they are phosphorylated. One possibility is that they become part of an ion current flux through the nuclear pores as a result of the added charge. Once inside the nucleus, they can then act as transcription factors, regulating the expression of their target genes. By modification of substrate activity, protein kinases also control many other cellular processes, including metabolism, transcription, cell cycle progression, cytoskeletal rearrangement and cell movement, apoptosis, and differentiation. Protein phosphorylation also plays a critical role in intercellular communication during development, in physiological responses, in homeostasis, and in the functioning of the nervous and immune systems. (Manning et al., 2002).
The crucial role that kinases play in biomolecular systems makes the enzymes important targets for academic and applied research. Fundamental issues to be addressed by current biomolecular investigations include: At what rate a given kinase marks a target protein? What are the target proteins of a given kinase? How do pharmaceuticals alter and regulate kinase activity? These questions are of great scientific and economic importance, as illustrated by the fact that kinase inhibitors are one of the largest classes of drugs on the market.
There have been many developments over the past few decades that hold great promise for future kinase research. One such achievement, the sequencing of the human genome, has provided an explosion of information that is now being analyzed and studied by the scientific community. Another highly fruitful area of scientific inquiry has been the emerging field of proteomics. Researchers in the field of proteomics are applying the genetic information elucidated by the human genome project and beginning to understand the functions of encoded proteins.
This burgeoning growth in genome and proteome investigation has been ushered in by a new wave of high-throughput assays and arrays that have enabled investigators to rapidly analyze and sequence enormous amounts of genetic data. One such high-throughput assay, the DNA microarray, is able to facilitate the rapid identification and classification of thousands of genes simultaneously. A DNA microarray works by exploiting the ability of a given mRNA molecule to bind specifically to, or hybridize to, the DNA template from which it originated. By using an array containing many DNA samples, scientists can determine, in a single experiment, the expression levels of thousands of genes within a cell by measuring the amount of mRNA bound to each site on the array. With the aid of computers and computational algorithms, the amount of mRNA bound to the spots on the microarray is precisely measured, generating a profile of gene expression in the cell. (NIH Primer on Microarrays, 2011).
Another such development, the protein microarray, holds promise as a research tool that will help scientists better understand the role that the encoded proteins of the human genome play within the intact biological system. The protein microarray can be constructed by immobilizing large numbers of purified proteins and used to assay a wide range of biochemical functions.
Despite the great advances in understanding that have been brought about by this new generation of high-throughput assay technology, there are still significant technological hurdles that are inherent in the assay technology itself. For instance, DNA and protein microarray technology is normally dependent upon an optical signal being generated upon binding of an analyte of interest, i.e. detection of a fluorescently-labeled nucleic acid tag. This dependency upon a fluorescently-labeled tag brings with it consequent restraints relating to the sensitivity, speed, cost, and miniaturization capabilities of these assays. A unique technological challenge facing protein microarray technology in particular, is caused by the sensitivity and heterogeneity of proteins, which make it difficult to stably store protein arrays in a functional state for long periods of time. In contrast, DNA is a highly stable molecule capable of long-term storage. Two recent advances may help researchers overcome these previous constraints.
The first, Ion-Sensitive Field-Effect Transistor chip technology (ISFET chips) is beginning to be conceptualized as a viable method for non-optical biosensing technology that holds promise as an alternative to the traditional microarrays that are dependent upon fluorescently labeled tags. ISFET chips generally work by measuring fluctuations in ion concentration (H+ or OH−) in a solution that contains an analyte of interest. Therefore, large ISFET chip arrays can be constructed and the variation in charge density of an analyte of interest contained in the various wells of the chip can be measured; thereby allowing for the electronic analysis of biomolecules. (Lee et al., 2009).
The second, Nucleic Acid-Programmable Protein Array (NAPPA) technology, addresses the stability problems inherent in constructing protein arrays. NAPPA technology replaces the complex process of spotting purified proteins with the simple process of spotting plasmid DNA. NAPPA exploits the ability of researchers to transfer protein encoding regions (open reading frames; ORFs) into specialized tagged expression vectors. These new expression clones are then spotted on the array and the proteins are then produced in situ in a cell-free system and immobilized in place upon their synthesis. This minimizes direct manipulation of the proteins and produces them just-in-time for the experiment, avoiding problems with protein purification and stability. (Ramachandran et al., 2004). NAPPA arrays have been developed that allow for thousands of proteins to be produced simultaneously in situ, and with remarkably consistent protein levels displayed. (Ramachandran et al., 2008). The power of this approach is that by expressing many proteins on a single array it is possible to test the function of many proteins simultaneously.
Despite the recent advances in high-throughput assay technology, there has not been adequate utilization of these methods and devices in the study of the human proteome and in particular the human kinome. The human kinome comprises 1.7% of all human genetic information and there are over 500 proteins encoded by these genes. These kinome proteins are well known for their importance in normal cell physiology and for their role in many human diseases. Consequently, drugs related to inhibiting kinase expression are one of the largest classes available on the market. It can therefore be easily seen that an understanding of kinase activity in living systems holds great promise for addressing a whole host of human diseases and genetic conditions. Therefore, there is a need in the arena of kinase research for the implementation of new methods and devices that take advantage of the rapidly developing advances in the field of high-throughput molecular biology assays.
For the foregoing reasons, there is a need for methods and devices that can take advantage of the recent technological advances seen in the electrical biosensing arena and the advances made with protein arrays.